Mehran Haidari

Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis

Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule–1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow–derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1–deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis.

Fasting and postprandial overproduction of intestinally-derived lipoproteins in an animal model of insulin resistance: Evidence that chronic fructose feeding in the hamster is accompanied by enhanced intestinal de novo lipogenesis and apoB48-containing lipoprotein overproduction

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (a)in vivo Triton WR1339 studies of apoB48 production as well as (b) ex vivo pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding orin vitro incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte de novo lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.

Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir

Influenza epidemics cause numerous deaths and millions of hospitalizations each year. Because of the alarming emergence of resistance to anti-influenza drugs, there is a need to identify new naturally occurring antiviral molecules. We tested the hypothesis that pomegranate polyphenol extract (PPE) has anti-influenza properties. Using real time PCR, plaque assay, and TCID 50% hemagglutination assay, we have shown that PPE suppresses replication of influenza A virus in MDCK cells. PPE inhibits agglutination of chicken red blood cells (cRBC) by influenza virus and is virucidal. The single-cycle growth conditions indicated that independent of the virucidal effect PPE also inhibits viral RNA replication. PPE did not alter virus ribonucleoprotein (RNP) entry into nucleus or translocation of virus RNP from nucleus to cytoplasm in MDCK cells. We evaluated four major Polyphenols in PPE (ellagic acid, caffeic acid, luteolin, and punicalagin) and demonstrated that punicalagin is the effective, anti-influenza component of PPE. Punicalagin blocked replication of the virus RNA, inhibited agglutination of chicken RBCs by the virus and had virucidal effects. Furthermore, the combination of PPE and oseltamivir synergistically increased the anti-influenza effect of oseltamivir. In conclusion, PPE inhibited the replication of human influenza A/Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.

Influenza virus directly infects, inflames, and resides in the arteries of atherosclerotic and normal mice

OBJECTIVE Influenza can trigger heart attacks, and vaccination against influenza reduces the risk of cardiovascular events. Currently, it is believed that influenza virus in general does not disseminate to extra-pulmonary tissues. We assessed the vascular effects of influenza infection and whether the virus can directly infect atherosclerotic arteries in mice. METHODS/RESULTS We intranasally infected 4 different types of mice--atherosclerotic apo E-deficient (our primary model), LDL receptor knockout, C57BL/6, and outbred Swiss--with influenza A/HK (H3/N2) virus. On day 7 after infection, we cultured viable virus from lung, aorta, and heart tissue, but not from the blood of apo E-deficient mice. Immunofluorescence studies showed influenza A virus NP1 protein and real time polymerase chain reaction (PCR) assay showed RNA in the aorta of infected apo E-deficient mice. Infected mice had significantly higher blood levels of chemokines and cytokines than control mice. At the local level, gene expression for several chemokines and cytokines was increased and eNOS expression was decreased. Infected mice had a higher density of macrophages in plaque than did control mice. CONCLUSIONS We have shown for the first time that influenza virus can directly infect and reside in atherosclerotic arteries and that infection was associated with systemic and arterial-level pro-inflammatory changes.

Evaluation of C-reactive protein, a sensitive marker of inflammation, as a risk factor for stable coronary artery disease.

OBJECTIVES Multiple lines of investigations have converged to suggest a prominent role for inflammation in coronary artery disease (CAD). The association of CRP level with active CAD is well documented. The relation, however, between levels of CRP and the presence and extent of stable CAD has seldom been studied in the developing countries. We investigated the association between serum concentration of C-reactive protein (CRP) and angiographically documented coronary artery disease (CAD) in a population of 450 individuals. DESIGN AND METHODS Ultrasensitive immunoassay was used to measure CRP levels in 284 patients with CAD and 166 control healthy subjects. The association of CRP levels with severity of disease as indicated by > or = 50% stenosis in one vessel (n = 79), two vessels (n = 74), or three vessels (n = 131) was also investigated. RESULTS CRP levels were greater in the patients with CAD (2.14 (0.88--3.38) vs. 1.45 (0.70--2.55) mg/L, p < 0.0001) than in the respective control subjects. Multiple logistic regression analysis showed CRP as an independent discriminating risk factor for CAD (odds ratio, 3.46, p < 0.001). Significant correlation was identified between CRP levels and severity of CAD (p < 0.0001). Prediction models that incorporated CRP in addition to other established coronary risk factors were significantly better at predicting risk than the models based on the other risk factors alone. CRP level was also an independent predictor of CAD in a subpopulation with normal levels of low density lipoprotein cholesterol (LDL-C < or = 3.4 mmol/L, p < 0.009). CONCLUSIONS Our findings suggest that CRP has a strong association with stable CAD, as such, the measurement of CRP may improve the coronary risk assessment in Iranian CAD patients.

Integrin α2β1 mediates tyrosine phosphorylation of vascular endothelial cadherin induced by invasive breast cancer cells.

Background: The disruption of endothelial barrier function by tumor cells was studied. Results: The attachment of tumor cells to endothelial cells leads to the disorganization of endothelial adherens junction. Conclusion: Interaction of tumor cells with endothelial cells alters endothelial signaling and facilitates cancer cell diapedesis. Significance: This study introduces new therapeutic targets for treating metastatic breast cancer. The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of β-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α2β1 integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α2β1 integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of β-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with β1-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α2β1 integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.

Increased oxidative stress in atherosclerosis-predisposed regions of the mouse aorta.

AIMS The localization of atherosclerotic lesions to predictable regions in mammalian arteries has been recognized for over a century. We sought to investigate the association between oxidative stress and regional susceptibility of the mouse aorta to atherosclerosis. MAIN METHODS En face confocal microscopy was employed to assess oxidative stress in the aortic intima of atherosclerosis-susceptible and protected regions of wild-type C57BL/6 mouse. Expression of reactive oxygen species and antioxidant producing genes were compared in endothelial cells from the susceptible and protected regions. KEY FINDINGS In vivo administration of redox-sensitive fluorescent dyes revealed an increase in the production of reactive oxygen species (ROS) in the atherosclerosis-susceptible regions relative to the protected regions. In contrast, Hoechst a redox-insensitive dye distributed evenly in the susceptible and protected regions. Accumulation of superoxide in the susceptible regions of the aorta was significantly blocked by the administration of Diphenyleneiodonium, a flavoprotein inhibitor. mRNA levels of superoxide-producing and scavenging enzymes were significantly increased in the regions predisposed to atherosclerosis. The regional difference in oxidative stress was at a lesser magnitude in BALB/c than the atherosclerosis-susceptible mouse (C57BL/6). SIGNIFICANCE Our study for the first time demonstrated an augmented oxidative stress in atherosclerosis-susceptible regions of the normal mouse aorta.

Myosin light chain phosphorylation facilitates monocyte transendothelial migration by dissociating endothelial adherens junctions

AIMS Transendothelial migration (TEM) of monocytes is a crucial step in inflammatory processes such as atherogenesis. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the dissociation of adherens junctions and the increased paracellular permeability of endothelial cells (ECs) that occur during TEM of monocytes. However, the underlying molecular mechanism has not been determined. We tested the hypothesis that the phosphorylation of myosin light chain (MLC) in ECs is crucial for the dissociation of adherens junctions during TEM of monocytes. METHODS AND RESULTS Using a combination of biochemical and cellular techniques, we provide evidence for the signal transduction pathways that regulate tyrosine phosphorylation of VE-cad in ECs after the attachment of monocytes. Our findings indicate that after interaction of integrins on THP-1 cells with adhesion molecules on ECs, the induction of the HRas\Raf\MEK\ERK signalling cascade leads to the phosphorylation of MLC. This results in the recruitment of Src to the VE-cad complex and tyrosine phosphorylation of VE-cad, which leads to dissociation of β-catenin from the VE-cad complex, formation of gaps between ECs, and enhancement of THP-1 cell TEM. CONCLUSION Our studies suggest that monocyte-induced phosphorylation of MLC in ECs enhances TEM of monocytes through dissociation of EC adherens junctions.

Oligonol a low molecular weight polyphenol of lychee fruit extract inhibits proliferation of influenza virus by blocking reactive oxygen species-dependent ERK phosphorylation

The emergence of resistance to anti-influenza drugs calls for the search for new antiviral molecules with different resistance profiles. Polyphenolic compounds are found in various plants and have antiviral and antioxidative properties. We tested the hypothesis that oligonol, a lychee fruit-derived low molecular weight polyphenol, possesses anti-influenza effects by inhibiting phosphorylation of extracellular-signal-regulated kinases (ERK). Real time PCR, plaque assay, and immunofluorescence techniques were used to study the effects of oligonol on proliferation of influenza virus. Oligonol inhibits influenza virus proliferation by blocking attachment of the virus to MDCK cells and by suppression of nuclear export of influenza virus ribonucleoprotein (RNP). Infection of MDCK cells with influenza virus leads to an increase in production of reactive oxygen species (ROS) and induction of a ROS-dependent ERK phosphorylation. Inhibition of ERK activation by a dominant negative mutant of ERK or N-acetyl-cysteine (NAC) leads to inhibition of influenza RNP nuclear export. Phorbol 12-myristate 13-acetate (PMA) induces ROS production, ERK phosphorylation and enhances influenza proliferation in MDCK cells. Oligonol and NAC inhibit PMA-induced ERK phosphorylation and ROS production. Our studies suggest that the underlying mechanism for the inhibitory effect of oligonol on influenza virus RNP nuclear export is blocking of ROS-dependent induction of ERK phosphorylation.

Apolipoprotein B as the best predictor of coronary artery disease in Iranian normolipidemic patients.

OBJECTIVES A relatively high proportion of Iranian patients with coronary artery disease (CAD) have normal levels of traditional lipid risk factors and show early onset of CAD. In this study we examined the roles of apolipoprotein B (apoB), apolipoprotein AI (apoAI) and lipoprotein (a) [LP(a)] in predicting coronary heart disease in normolipidemic patients and those with premature CAD (age < or = 50). DESIGN AND METHODS Serum levels of apoB, apoAI, and LP(a) were determined in a total of 567 Iranian patients who were candidates for coronary angiography. A subgroup of 142 patients (93 males, 49 females) with normal levels of classical lipid risk factors, and a subgroup of patients (130 males, 71 females) with age below 50 years were separately assessed for coronary risk factors. RESULTS ApoB concentrations were significantly higher in patients with CAD (CAD+) relative to patients without CAD (CAD-) in the two subgroups. Multiple logistic regression after controlling for age and others risk factors showed apoB as the best determinant of CAD in the normolipidemic subgroup (OR, 4.3, p < 0.001) and in the men aged < or = 50 (OR, 5.7, p < 0.001). ApoB was the best predictor of CAD in a subgroup of very young patients (age < or = 40, n = 77, OR, 8.6, p < 0.009). There was a significant correlation between severity of atherosclerosis and serum apoB concentration in the normolipidemic subgroup (r = 0.22, p < 0.008). CONCLUSIONS Our data indicate that serum concentration of apoB is the best discriminating factor to predict the presence or absence of atherosclerosis in Iranian normolipidemic individuals and young patients undergoing coronary angiography.